Malaria caused by Plasmodium falciparum is a leading cause of mortality and morbidity in the inter-tropical regions of the world and is worsening as a result of resistance to existing classes of antimalarials. Recent work has demonstrated the existence in P. falciparum of a fatty acid synthase type-II (FAS-II) pathway that constitutes a core function of the parasite apicoplast, a unique organelle whose inhibition leads to parasite death. The absence of the FAS-II pathway in humans and the identification of specific FAS-II antimalarial inhibitors, including triclosan that targets enoyl ACP reductase, validate this pathway in general and P. falciparum enoyl ACP reductase (PfENR) in particular as outstanding targets for the development of new antimalarial drugs. We propose a 5-year program to carry out a high throughput screen for PfENR inhibitors and develop at least two chemical series into a candidate antimalarial for preclinical development. Our Project 3 proposes molecular genetic approaches to improve the in vivo screening for potent Plasmodial ENR inhibitors and to screen for resistance. In Aim 1, we will implement molecular genetic techniques to develop transgenic P. berghei rodent malaria parasites that express pfenr or pvenr, the target of the two most important human malaria parasites, in the place of the rodent malarial enzyme, to improve the predictive power of the rodent in vivo screening. In Aim 2, we will develop bacterial and parasite model systems to screen for resistance to PfENR inhibitors. The bacterial system will use E. coli lines that express temperature-sensitive forms of FabI (the E. coli ortholog of PfENR) that can be complemented by PfENR. This will enable us to select for resistance to ENR inhibitors at temperatures at which only PfENR can function. For selected compounds with good in vitro and in vivo potency profiles, we will also screen directly for resistance. These resistant PfENR alleles will be analyzed biochemically and structurally in Project 1, thus providing valuable information for further chemical refinement of suitable lead compounds.